The present invention is directed to compositions comprising hapten-modified tumor cells and extracts and methods of treating cancer by administering a therapeutically effective amount of a composition comprising a tumor cell or tumor cell extract to a subject in need of such treatment. The invention also relates to an effective vaccination schedule useful for inducing an antitumor response in a patient suffering from cancer.
It was theorized in the 1960""s that tumor cells bear specific antigens (TSA) which are not present on normal cells and that the immune response to these antigens might enable an individual to reject a tumor. It was later suggested that the immune response to TSA could be increased by introducing new immunological determinants on cells. Mitchison, Transplant. Proc., 1970, 2, 92. Such a xe2x80x9chelper determinantxe2x80x9d, which can be a hapten, a protein, a viral coat antigen, a transplantation antigen, or a xenogenous cell antigen, could be introduced into a population of tumor cells. The cells would then be injected into an individual who would be expected to be tolerant to the growth of unmodified tumor cells. Clinically, the hope was that an immunologic reaction would occur against the helper determinants, as a consequence of which the reaction to the accompanying TSA is increased, and tumor cells which would otherwise be tolerated are destroyed. Mitchison, supra, also suggests several modes of action of the helper determinants including 1) that the unmodified cells are merely attenuated, in the sense that their growth rate is slowed down or their susceptibility to immunologic attack increased; 2) that helper determinants merely provide points of attack and so enable the modified cells to be killed by an immune response not directed against TSA; 3) that the helper determinants have an adjuvant action such as binding to an antibody or promoting localization of the cells in the right part of the body for immunization, in particular, in lymph nodes.
Fujiwara et al., J. Immunol., 1984, 132, 1571 showed that certain haptenized tumor cells, i.e., tumor cells conjugated with the hapten trinitrophenyl (TNP), could induce systemic immunity against unmodified tumor cells in a murine system, provided that the mice were first sensitized to the hapten in the absence of hapten-specific suppressor T cells. Spleen cells from the treated mice completely and specifically prevented the growth of tumors in untreated recipient animals. Flood et al;, J. Immunol., 1987, 138, 3573 showed that mice immunized with a TNP-conjugated, ultraviolet light-induced xe2x80x9cregressorxe2x80x9d tumor were able to reject a TNP-conjugated xe2x80x9cprogressorxe2x80x9d tumor that was otherwise non-immunologic. Moreover, these mice were subsequently resistant to challenge with unconjugated xe2x80x9cprogressorxe2x80x9d tumor. In another experimental system, Fujiwara et al., J. Immunol., 1984, 133, 510 demonstrated that mice sensitized to trinitrochlorobenzene (TNCB) after cyclophosphamide pretreatment could be cured of large (10 mm) tumors by in situ haptenization of tumor cells; subsequently, these animals were specifically resistant to challenge with unconjugated tumor cells.
The teachings of Fujiwara et al. differ from the present invention for several reasons including the following: A. The cells used in Fujiwara""s composition are derived from induced transplantable murine tumorsxe2x80x94not from spontaneous human tumors; B. Fujiwara""s composition is used in immunoprophylaxisxe2x80x94the present invention uses immunotherapy; C. Fujiwara""s composition is administered as a local therapyxe2x80x94the composition of the present invention is administered by systemic inoculation; and D. Fujiwara""s composition did not result in tumor regressionxe2x80x94the composition of the present invention results in regression and/or prolonged survival for at least a substantial portion of the patients treated.
The existence of T cells which cross-react with unmodified tissues has recently been demonstrated. Weltzien and coworkers have shown that class I MHC-restricted T c6ell clones generated from mice immunized with TNP-modified syngeneic lymphocytes respond to MHC-associated, TNP-modified xe2x80x9cselfxe2x80x9d peptides. Ortmann, B.,: et al., J. Immunol., 1992, 148, 1445. In addition, it has been established that immunization of mice with TNP-modified lymphocytes results in the development of splenic T cells that exhibit secondary proliferative and cytotoxic responses to TNP-modified cells in vitro. Shearer, G. M. Eur. J. Immunol., 1974, 4, 527. The potential of lymphocytes elicited by immunization with DNP- or TNP-modified autologous cells to respond to unmodified autologous cells is of considerable interest because it may be relevant to two clinical problems: 1) drug-induced autoimmune disease, and 2) cancer immunotherapy. In regard to the former, it has been suggested that ingested drugs act as haptens, which combine with normal tissue protein forming immunogenic complexes that are recognized by T cells. Tsutsui, H., et al., J. Immunol., 1992, 149, 706. Subsequently, autoimmune disease, e.g., systemic lupus erythematosus, can develop and continue even after withdrawal of absence of the offending drug. This would imply the eventual generation of T lymphocytes that cross-react with unmodified tissues.
The common denominator of these experiments is sensitization with hapten in a milieu in which suppressor cells are not induced. Spleen cells from cyclophosphamide pretreated, TNCB-sensitized mice exhibited radioresistant xe2x80x9camplified helper functionxe2x80x9d i.e., they specifically augmented the in vitro generation of anti-TNP cytotoxicity. Moreover, once these amplified helpers had been activated by in vitro exposure to TNP-conjugated autologous lymphocytes, they were able to augment cytotoxicity to unrelated antigens as well, including tumor antigens (Fujiwara et al., 1984). Flood et. al., (1987), supra, showed that this amplified helper activity was mediated by T cells with the phenotype Lytxe2x88x921+, Lytxe2x88x922xe2x88x92, L3T4+, Ixe2x88x92J+ and suggests that these cells were contrasuppressor cells, a new class of immunoregulatory T cell.
Immunotherapy of patients with melanoma had shown that administration of cyclophosphamide, at high dose (1000 mg/M2) or low dose (300 mg/M2), three days before sensitization with the primary antigen keyhole limpet hemocyanin markedly augments the acquisition of delayed type hypersensitivity to that antigen (Berd et al., Cancer Res., 1982, 42, 4862; Cancer Res., 1984, 44, 1275). Low dose cyclophosphamide pretreatment allows patients with metastatic melanoma to develop delayed type hypersensitivity to autologous melanoma cells in response to injection with autologous melanoma vaccine (Berd et al., Cancer Res., 1986, 46, 2572; Cancer Invest., 1988, 6, 335). Cyclophosphamide administration results in reduction of peripheral blood lymphocyte non-specific T suppressor function (Berd et al., Cancer Res., 1984, 44, 5439; Cancer Res., 1987, 47, 3317), possibly by depleting CD4+, CD45R+ suppressor inducer T cells (Berd et al., Cancer Res., 1988, 48, 1671). The anti-tumor effects of this immunotherapy regimen appear to be limited by the excessively long interval between the initiation of vaccine administration and the development of delayed type hypersensitivity to the tumor cells (Berd et al., Proc. Amer. Assoc. Cancer Res., 1988, 29, 408 (#1626)). Therefore, there remains a need to increase the therapeutic efficiency of such a vaccine to make it more immunogenic.
Most tumor immunologists now agree that infiltration of T lymphocytes, white cells responsible for tumor immunity, into the tumor mass is a prerequisite for tumor destruction by the immune system. Consequently, a good deal of attention has been focused on what has become known as xe2x80x9cTILxe2x80x9d therapy, as pioneered by Dr. Stephen Rosenberg at NCI. Dr. Rosenberg and others have extracted from human cancer metastases the few T lymphocytes that are naturally present and greatly expanded their numbers by culturing them in vitro with Interleukin 2 (IL2), a growth factor for T lymphocytes. Topalian et al., J. Clin. Oncol., 1988, 6, 839. However this therapy has not been very effective because the injected T cells are limited in their ability to xe2x80x9chomexe2x80x9d to the tumor site.
The ability of high concentrations of IL2 to induce lymphocytes to become non-specifically cytotoxic killer cells has been exploited therapeutically in a number of studies (Lotze et al., J. Biol. Response, 1982, 3, 475; West et al., New Engl. J. Med., 1987, 316, 898). However, this approach has limitations due to the severe toxicity of high dose intravenous IL2. Less attention has been given to the observation that much lower concentrations of IL2 can act as an immunological adjuvant by inducing the expansion of antigen-activated T cells (Talmadge et al., Cancer Res., 1987, 47, 5725; Meuer et al., Lancet, 1989, 1, 15). Therefore, there remains a need to understand and attempt to exploit the use of IL2 as an immunological adjuvant.
Human melanomas are believed to express unique surface antigens recognizable by T lymphocytes. Old, L. J., Cancer Res., 1981, 41, 361; Van der Bruggen, P., et al., Science, 1991, 254, 1643; Mukherji, B., et al., J. Immunol., 1986, 136, 1888; and Anichini, A., et al., J. Immunol., 1989, 142, 3692. However, immunotherapeutic: approaches prior to work done by the present inventor had been limited by the difficulty of inducing an effective T cell-mediated response to such antigens in vivo.
There are several models proposed to explain what appears to be tolerance to human tumor-associated antigens. They include:
1) Tumor antigen-specific suppressor cells that down-regulated incipient anti-tumor responses. Mukherji, et al., supra; Berendt, M. J. and R. J. North., J. Exp. Med., 1980, 151, 69.
2) Failure of human tumor cells to elicit T helper cells or to provide costimulatory signals to those T cells. Fearon, E. R., et al., Cell, 1990, 60, 397; Townsend, S. E. and J. P. Allison, Science, 1993, 259, 368; and
3) Reduced surface expression of major histocompatibility products: on tumor cells which limits their recognition by T cells. Ruiter, D. J., Seminars in Cancer Biology, 1991, 2, 35. None of these hypotheses has yet been corroborated in a clinical system.
The goal of active immunotherapy for tumors is the development of a productive systemic T cell mediated tumor-specific immunity. Tumor specific immunity would act both at the primary tumor site as well as in clearing small metastatic foci at distant sites. The generation of T cell immunity has been shown to be a highly regulated response requiring cell-cell interaction and the production of a number of cytokines. Of late, studies in a number of human and urine systems have shown that T cell responses can be separated into two categories termed Type I and II (Mossman, et al., J. Immunol. 1986 136:2348). Type I responses are required for the development of delayed type hypersensitivity (DTH), are associated with macrophase activation and the production of interferon-gamma (IFNxcex3), and have been shown to be associated with the resolution of human leprosy (Yamamura, M., et al., Science 1991 254:277-279) and murine leishmaniasis (Scott, P., et al., Immunological Review 1989 112:161-182). Type II responses are associated with the production of IL4 and IL10, primarily support antibody responses, and are associated with the progressive forms of leprosy (Yamamura, M., et al., supra) and leishmaniasis (Yamamura et al., supra; and Scott, P., et al., supra.). In addition to the development of DTH, Type I responses would be expected to enhance the generation of tumor specific CTL via upregulation of MHC and tumor associated antigens as well as enhanced antigen presentation secondary to localized IFNxcex3 production. More recently, Type I and II response have been shown to be cross regulating: IFNxcex3 inhibits Type II responses, while IL4 and IL10 inhibit Type I (Scott, J. Immunol. 1991 147:3149-3155; and Fiorentino et al., J. Immunol. 1991 146:3444-3451). In the leishmania system, modulation of cytokines at the lesion site allows for the conversion of a Type II to a Type I response, and, consequently, a change from progressive infection to eradication of the disease (Scott, 1991, supra).
Pisa et al., Proc. Natl. Acad. Sci. USA 1992 89:7708-7712 detected IL10 mRNA in ovarian carcinoma biopsies, but not in ovarian carcinoma cell lines; they concluded that the source of IL10 was tumor-infiltrating lymphocytes. Gastl et al, Int. J. Cancer 1991 55:96-101 found that 16/48 tumor cell lines released IL10 into the culture supernatant; only 3-8 melanoma cell lines were positive. Finally, Chen et al., Int. J. Cancer, 1994 56:755-760 recently reported that 6/9 cell lines derived from metastatic melanomas expressed IL10 mRNA. However, the present invention is the first report known to the inventors of mRNA for IL10 in metastatic melanoma biopsies.
It is not known whether these observations are applicable to the human tumor-host relationship, i.e., whether the pattern of cytokine production by T cells infiltrating tumors is an indicator of the effectiveness of the immune response. Patients with metastatic melanoma treated with an autologous, DNP-modified vaccine develop inflammatory responses at tumor sites, Berd et al., 1991, supra. Histologically, these inflamed lesions are characterized by T cell infiltration which is sometimes associated with tumor cell destruction. The present invention finds that tumors from DNP-vaccine-treated patients contain Type I T lymphocytes, which are not detectable in tumors excised prior to vaccine administration.
Conventional attempts to treat human cancer have been unsuccessful or only partially successful, and often have undesirable side effects. Attempts to treat cancer based on various immunological theories have also been unsuccessful. Although the Applicant has successfully treated melanoma in certain patients using hapten-conjugated melanoma cells, there remains a need in the cancer treatment art for additional and improved methods for inducing an anti-tumor response. Applicant has now discovered an effective vaccination protocol using hapten-modified tumor cells or extracts.
The present invention relates to compositions containing hapten-modified tumor cells and tumor cell extracts and methods for inducing an antitumor response in a patient suffering from cancer by administering the compositions of the invention.
According to one aspect, the present invention relates to an isolated mammalian, preferably human, tumor cell or tumor cell extract modified with a hapten.
In another aspect, the present invention is directed to a composition comprising a hapten modified mammalian tumor cell or extract.
In yet another aspect, the invention provides for a vaccine composition comprising a therapeutically effective amount of a mammalian, preferably human, tumor cell or extract modified with a hapten.
In yet another aspect, the present invention is directed to a method of treating cancer comprising administering to a mammal, preferably a human, a composition comprising a therapeutically effective amount of a hapten modified human tumor cell or extract wherein said mammal suffers from a malignant tumor of the same type as said tumor cell membrane.
In a further aspect, the invention is directed to a method of treating cancer according to a weekly vaccination schedule.